Lyophilized preparation of cytotoxic dipeptides

ABSTRACT

The present invention is directed to novel lyophilized pharmaceutical preparations comprising a cytotoxic dipeptides such as melphalan flufenamide and one or more excipient(s) selected from the group comprising a polysorbate; a polyethylene glycol; β-cyclodextrin; ocyclodextrin; hydroxypropyl-β-cyclodextrin; sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodium succinate; propylene glycol; Cremophor EL; Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose and an amino acid. This preparation may be further formulated and is useful in cancer therapy.

This application is a continuation of U.S. application Ser. No.14/113,768, which is a national stage application of internationalapplication no. PCT/EP2012/057577, filed Apr. 25, 2012, which claims thepriority benefit of U.S. provisional application No. 61/535,126, filedSep. 15, 2011, and Swedish application no. 1150371-1, filed Apr. 28,2011, the contents of each of which are incorporated by reference hereinin their entireties.

TECHNICAL FIELD

The present invention is directed to lyophilized pharmaceuticalpreparations comprising cytotoxic dipeptides or pharmaceuticallyacceptable salts thereof, methods for their preparation, compositionscomprising the lyophilized pharmaceutical preparations and their use inthe treatment of cancer.

BACKGROUND ART

Cancer is a disease which is difficult to cure and which may be fatal.Accordingly, efforts to develop new therapies for cancer are constantlyongoing in the research society. The vast majorities of cancers arepresent as solid tumors, e.g. lung cancer, breast cancer, prostatecancer, while the rest are hematological and lymphoid malignancies, e.g.leukemias and lymphomas.

Chemotherapy is often used in attempts to cure or palliate the disease.As cancer cells typically divide rapidly, chemotherapy usually acts bykilling rapidly dividing cells. In the broad sense, mostchemotherapeutic drugs work by impairing mitosis (i.e. cell division),effectively targeting fast-dividing cells. As these drugs cause damageto cells they are termed cytotoxic. Some drugs cause cells to undergoapoptosis (so-called “programmed cell death”). Often combinationchemotherapy is used, when two or more drugs having different modes ofaction are used together in order to optimise the antitumoral effect, tominimise side effects, and prevent resistance development. The resultsobtained with chemotherapy vary according to tumor type. Some tumors arevery sensitive and the treatment has then a high probability of leadingto cure.

Chemotherapeutic drugs can generally be divided into alkylating agents,antimetabolites, anthracyclines, plant alkaloids, topoisomeraseinhibitors, and other antitumor agents. The drugs affect cell divisionor DNA synthesis.

Alkylating agents, such as drugs derived from nitrogen mustard, that isbis(2-chloroethyl)amine derivatives, are used as chemotherapeutic drugsin the treatment of a wide variety of neoplastic diseases. Alkylatingagents have the ability to covalently attach alkyl groups toelectronegative sites in cells. Thus, these agents act by impairing cellfunction by forming covalent bonds with heteroatoms in biologicallyimportant molecules like RNA, DNA and proteins. Examples of alkylatingagents are mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide,temozolomide and melphalan that chemically modify a cell's DNA.

WO01/96367 discloses alkylating di- and tripeptides and one or twoadditional amino acids or amino acid derivatives. These derivatives weredemonstrated to have an improved efficacy on a variety of tumor types.

Melphalan, i.e. p-[bis-(2-chloroethyl)amino]phenylalanine, is aconjugate of nitrogen mustard and the amino acid phenylalanine, whichwas synthesised in the mid 1950s (U.S. Pat. No. 3,032,584). This classicalkylating substance soon became a valuable drug in the chemotherapeuticfield and is still of importance in the treatment of for examplemyeloma. Clinical use of melphalan in the treatment of late stage solidtumors has, however, had limited efficacy. In the search for a moreselective action on malignant cells melphalan analogues have thereforebeen synthesised.

Larionov L. F., Cancer Res (1961), 21, 99-104 discloses variousmelphalan-related derivatives.

STN registry files RN: 1060633-95-5, RN: 887609-28-1, RN 790650-89-4,RN: 781606-39-1, RN: 773046-98-3, RN: 767621-58-9, RN: 760165-58-0 andRN: 757941-61-0 discloses various melphalan-related derivatives.

Koltun, M et al., Biopharmaceutics & Drug disposition (210), 31, 450-454discloses forms of melphalan.

Ma D Q et al., International Journal of Pharmaceutics (1999), 189,227-234 discloses discloses forms of melphalan.

Murav'ev I et al., Farmatsiya (1978), 27, (2), 13-15 (with abstract inChemical Abstracts no. 1978:412066) discloses melphalan-relatedderivatives.

Lyophilization or freeze-drying is a method for dehydrating samples usedto preserve or increase stability or to stop degradation. Due to the lowwater content of lyophilized products, typically around 1-4%, the actionof microorganisms and enzymes is inhibited and the product life therebyincreased. In lyophilization, the sample to be lyophilized is dissolvedin an aqueous solution and subsequently frozen after which thesurrounding pressure is reduced. The sample is then submitted tosublimation, optionally by the application of heat, in order to sublimethe frozen water directly from the solid phase to the gas phase. Thefinal water content in the product is very low, typically around 1% to4%. Lyophilization is commonly used in the pharmaceutical field in orderto increase the shelf life of pharmaceutical products.

SUMMARY OF THE INVENTION

In general, lipophilic dipeptide ester derivatives suffer from a poorsolubility in aqueous solutions. Therefore, the use of organic solvents,such as DMA (dimethylacetamide), is necessary in order to dissolve suchdipeptides. However, organic solvents are often toxic and may also causedestruction of medical devices used for the administration of thedipeptides to subjects, such as cancer patients. Consequently, toovercome the problems with dissolving and providing the cytotoxicdipeptides in an organic solvent, there is a need for alternativepharmaceutical preparations of cytotoxic dipeptides having sufficientsolubility in physiologically acceptable solutions.

The present invention refers to lyophilized preparations comprisingmelphalanyl-L-p-fluorophenylalanine ethyl ester, also known as melphalanflufenamide, as well as pharmaceutically acceptable salt thereof, inparticularly, melphalanyl-L-p-fluorophenylalanine ethyl esterhydrochloride, also known as melphalan flufenamide hydrochloride, or J1.

An aspect of the present invention is directed to a lyophilizedpharmaceutical preparation comprising

(i) melphalan flufenamide, or a pharmaceutically acceptable saltthereof; and(ii) at least one excipient selected from the group comprising

-   -   a polysorbate; a polyethylene glycol; β-cyclodextrin;        α-cyclodextrin; hydroxypropyl-β-cyclodextrin;        sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol;        disodium succinate; propylene glycol; Cremophor EL; Dimethyl        sulfoxide; D-mannitol; Trehalose; Sucrose and an amino acid.

Still an aspect of the present invention is a lyophilized pharmaceuticalpreparation which is soluble in an aqueous solution.

Yet an aspect of the present invention is a method for the preparationof a lyophilized pharmaceutical preparation as herein described,whereby:

-   -   a. melphalan flufenamide, or a pharmaceutically acceptable salt        thereof, is dissolved in an organic solvent to obtain a        melphalan flufenamide solution;    -   b. water is added to the melphalan flufenamide solution in order        to obtain an aqueous melphalan flufenamide solution, in a        concentration of about 0.2-3.0 mg/ml;    -   c. at least one excipient selected from the group comprising a        polysorbate;        -   a polyethylene glycol; β-cyclodextrin; α-cyclodextrin;            hydroxypropyl-β-cyclodextrin;            sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol;            disodium succinate; propylene glycol; Cremophor EL; Dimethyl            sulfoxide; D-mannitol; Trehalose; Sucrose and an amino acid            is added to the melphalan flufenamide solution; and    -   d. the aqueous melphalan flufenamide solution containing        excipient(s) is subjected to lyophilization.

Still an aspect of the invention is a kit of parts, comprising a firstcontainer comprising a lyophilized pharmaceutical preparation as definedherein, and a second container comprising a physiologically acceptablesolution.

Still an aspect of the present invention is a lyophilized pharmaceuticalpreparation as herein described, for use as a medicament.

Yet an aspect of the invention is a kit of parts as herein described,for use as a medicament.

An aspect of the present invention is a lyophilized pharmaceuticalpreparation as herein described, for use in the treatment and/orprevention of cancer, such as ovarian cancer, lung cancer, bladdercancer, mesothelioma, multiple myeloma, breast cancer, and/or any solidor hematological cancer.

Yet an aspect of the invention is a kit of parts as herein described,for use in the treatment and/or prevention of cancer, such as ovariancancer, lung cancer, bladder cancer, mesothelioma, multiple myeloma,breast cancer, and/or any solid or hematological cancer.

Still an aspect of the present invention is a method for the treatmentof and/or prevention of cancer, such as ovarian cancer, lung cancer,bladder cancer, mesothelioma, multiple myeloma, breast cancer, and/orany solid or hematological cancer, whereby a lyophilized pharmaceuticalpreparation as described herein, is administered in a therapeuticallyeffective dose to a subject in need thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will supercede. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, drawings, examples, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-D contain graphs of four repeated dissolution speedmeasurements of melphalan flufenamide lyophilized without excipients bymethod A according to Example 2. Samples were withdrawn at the indicatedtime points and the amount of dissolved melphalan flufenamide wasdetermined by HPLC. The y-axis shows the amount of melphalan flufenamidein mg/ml.

FIGS. 2A-E contain graphs of dissolution speed measurements of melphalanflufenamide lyophilized in the presence of excipients as indicated inthe figures by method A according to Example 2. Samples were withdrawnat the indicated time points and the amount of dissolved melphalanflufenamide was determined by HPLC. The y-axis shows the amount ofmelphalan flufenamide in mg/ml.

FIG. 3 is a graph of dissolution speed measurement of melphalanflufenamide without excipients by method B according to Example 2.Samples were withdrawn at the indicated time points and the amount ofdissolved melphalan flufenamide was determined by HPLC. The y-axis showsthe amount of melphalan flufenamide in mg/ml.

FIGS. 4A-E contain graphs of dissolution speed measurements of melphalanflufenamide lyophilized in the presence of excipients as indicated inthe figures by method B. Samples were withdrawn at the indicated timepoints and the amount of dissolved melphalan flufenamide was determinedby HPLC. The y-axis shows the amount of melphalan flufenamide in mg/ml.

FIGS. 5A-D contain graphs of dissolution speed measurements as follows,A: melphalan flufenamide lyophilized without Polysorbate 80; B melphalanflufenamide lyophilized in the presence of 10% Polysorbate 80; Cmelphalan flufenamide lyophilized in the presence of 50% Polysorbate 80;D melphalan flufenamide lyophilized in the presence of 100% Polysorbate80. Amounts are relative to the amount of melphalan flufenamide. They-axis shows the amount dissolved melphalan flufenamide relative to theinternal standard as determined using HPLC.

FIG. 6 is a photograph of glass tubes with melphalan flufenamide (J1)that following lyophilization is dissolved in a concentration of 1 mg/mlin a 5% glucose solution containing 50% (mol) Polysorbate 80 (left) andno Polysorbate 80 (right).

FIGS. 7A-C contain structural formulas for melphalan flufenamide(L-melphalanyl-L-p-fluorophenylalanine ethyl ester) (J1),L-melphalanyl-L-p-fluorophenylalanine isopropyl ester (JV28), andL-prolinyl-L-melphalanyl-L-p-fluorophenylalanine ethyl ester (J3),respectively.

DETAILED DESCRIPTION OF THE INVENTION

Non-lyophilized cytotoxic dipeptides or pharmaceutically acceptablesalts thereof may have a low solubility in aqueous solutions, which maynecessitate the use of organic solvents, such as DMA(dimethylacetamide), for dissolving said dipeptides or pharmaceuticallyacceptable salts thereof. Therefore, when a cytotoxic dipeptide is to beadministered to a patient, the substance first has to be dissolved in anorganic solvent, such as DMA, and thereafter diluted in a solution forinfusion before administration to the patient. The patient is by thismethod exposed to organic solvents, the exposure of which may behazardous for the patient. Also, the organic solvent may destroy themedical devices used for the administration of melphalan flufenamide tosubjects, such as cancer patients.

The present inventors have now surprisingly found that when certaincytotoxic dipiptides or pharmaceutically acceptable salts thereof arelyophilized in the presence of an excipient, the resulting lyophilizedpharmaceutical preparation can have an even higher solubility in aphysiologically acceptable solution. In fact, the solubility can be sohigh that the step of dissolving the cytotoxic dipeptide orpharmaceutically acceptable salt thereof in an organic solvent can beomitted and the cytotoxic dipeptide can be directly dissolved in anaqueous, physiologically acceptable solution and administered to apatient. Preferably, said cytotoxic dipeptide is melphalan flufenamideor a pharmaceutically acceptable salt thereof.

In previous preparations, melphalan flufenamide was obtained fromsynthesis as a white powder in crystalline form. This crystalline formcan only be dissolved in highly acidic aqueous solutions, which forpractical manufacturing purposes is impossible. The presence ofexcipients as such, did not sufficiently improve the solubility.Therefore, previously melphalan flufenamide was instead dissolved in DMA(dimethylacetamide) in a glucose solution. The preparation is feasiblebut is unstable: 7% degradation/h. Furthermore, dimerization occurs andthe solution turns bright yellow. This preparation was, however,unreliable and the polymerization rate varied in an unacceptable manner.

Consequently, there is a need for identifying alternative ways ofproviding a preparation comprising melphalan flufenamide or apharmaceutically acceptable salt thereof that is soluble with increasedstability. Further, the preparation should be water-soluble to avoidnegative issues of having an organic solvent in the product that isprovided to the patient (such as DMA).

An aspect of the present invention is a lyophilized pharmaceuticalpreparation comprising

-   (i) melphalan flufenamide, or a pharmaceutically acceptable salt    thereof; and-   (ii) at least one excipient selected from the group comprising a    polysorbate; a polyethylene glycol; β-cyclodextrin; α-cyclodextrin;    hydroxypropyl-β-cyclodextrin; sulfobutylether-β-cyclodextrin;    lactose; benzyl alcohol; disodium succinate; propylene glycol;    Cremophor EL; Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose;    and an amino acid.

In one embodiment of this aspect, said excipient is selected from thegroup comprising Polysorbate 80; PEG 400; lactose; benzyl alcohol;disodium succinate; propylene glycol; PEG 300; Cremophor EL; Dimethylsulfoxide; D-mannitol; Trehalose; Sucrose; and histidine.

In another embodiment of this aspect, said melphalan flufenamide ismelphalan flufenamide hydrochloride (J1).

In another aspect of the invention, there is provided a pharmaceuticalpreparation comprising

-   (i) melphalan flufenamide hydrochloride (J1); and-   (ii) at least one excipient selected from the group comprising    -   a polysorbate; a polyethylene glycol; β-cyclodextrin;        α-cyclodextrin; hydroxypropyl-β-cyclodextrin;        sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol;        disodium succinate; propylene glycol; Cremophor EL; Dimethyl        sulfoxide; D-mannitol; Trehalose; Sucrose; and an amino acid.

In one embodiment of this aspect, said at least one excipient is apolysorbate or polyethylene glycol.

In another embodiment of this aspect, said at least one excipient isPolysorbate 80.

In another embodiment of this aspect, said at least one excipient hassurfactant properties. Such properties would increase the stability ofthe lyophilized pharmaceutical preparation. Said at least one excipienthaving surfactant properties may be polysorbate or polyethylene glycol,such as Polysorbate 80 or PEG400.

In another embodiment of this aspect the preparation comprises melphalanflufenamide hydrochloride (J1) and the excipient Polysorbate 80. Thepresence of the excipient Polysorbate 80 would increase the stability ofthe lyophilized pharmaceutical preparation. Further, the finalpreparation would be free, or essentially free of organic solvents, andtherefore less toxic.

The invention provides a lyophilized preparation which is stable in dryform and soluble in an aqueous solution without presence of an organicsolvent. While it previously was possible to prepare a lyophilizedpreparation of melphalan flufenamide alone, this preparation dissolvedtoo slowly in aqueous solutions compared to the degradation time.Incorporation of an excipient in the lyophilized melphalan flufenamidepreparation (via initial solution in an organic solvent) improves thereconstitution time considerably, but does not significantly alter thestability of reconstituted melphalan flufenamide. As a result, the timewindow for the reconstituted melphalan flufenamide is widened, and thisimproves the treatments of patients, e.g. by allowing for lower infusionrates, where needed. A preparation “without presence of an organicsolvent” could include trace amounts of organic solvent, typically lessthan 0.5% (w/w).

The lyophilized pharmaceutical preparation of melphalan flufenamide or apharmaceutically acceptable salt thereof as described herein, is awhite, fluffy powder in contrast to a non-lyophilized melphalanflufenamide or a pharmaceutically acceptable salt thereof, which can bein the form of a dense, slightly yellowish powder.

Typically, lyophilization comprises four steps, pretreatment, freezing,primary drying, and secondary drying. In the pretreatment step, thesubstance to be lyophilized is made ready for the lyophilization e.g. bypreparing a solution having the desired concentration or mixing thesubstance with further components in order to obtain an acceptableresult. The freezing step may be performed in a freeze-drying flask in abath cooled e.g. by mechanical refrigeration, dry ice and methanol, orliquid nitrogen. Freeze-drying machines are available for lyophilizationin a larger scale. Usually, the freezing temperatures are between −50°C. and −80° C.

In the primary drying step, the pressure is lowered to the range of afew millibars, and heat may be supplied for the water to sublimate fromthe material. The amount of heat necessary can be calculated using thesublimating molecules' latent heat of sublimation. The duration of thisperiod depends, but may last for days in order to preserve the materialsstructure.

The aim of the final secondary drying step is to remove any unfrozenwater molecules. In this phase, the temperature may be as high as above0° C., to break any physico-chemical interactions that have formedbetween the water molecules and the frozen material.

In the context of the present invention, it is to be understood thatmelphalan flufenamide or a pharmaceutically acceptable salt thereof, islyophilized. The term “a lyophilized pharmaceutical preparation of amelphalan flufenamide or a pharmaceutically acceptable salt thereof”, istherefore understood to mean that the melphalan flufenamide or apharmaceutically acceptable salt thereof is lyophilized.

Further aspects of the present invention provide lyophilized melphalanflufenamide or a pharmaceutically acceptable salt thereof, a kit ofparts comprising such melphalan flufenamide, methods for the preparationof such melphalan flufenamide or a pharmaceutically acceptable saltthereof, compositions comprising such lyophilized melphalan flufenamideor a pharmaceutically acceptable salt thereof and uses thereof.

“Lyophilization”, “lyophilized” etc. may in the present context be usedinterchangeably with “freeze-drying”, “freeze-dried” etc.

Examples of cytotoxic dipeptides that can be lyophilized as describedherein are set forth in WO01/96367. The N-terminus of a molecule shouldpreferably not be protected as amide or carbamate. This means that R₄ informula I therein should preferably not be a protecting group, such asformyl, acetyl or propionyl, or benzoyl, as the protected form of thecompound in general has a lower cytotoxic activity than thecorresponding free form. Natural amino acids refer to amino acids thatare normally existing and exerting their functions in living organisms.Modified amino acids refer to amino acids that in some way have beenmodified into a different chemical structure and chemical compositionthan a natural amino acid. An example of a natural cyclic amino acid isproline. Examples of aromatic amino acids are phenylalanine, tyrosine,tryptophan, and histidine.

The cytotoxic dipeptides, such as melphalan flufenamide, may alsocontain unnatural proportions of atomic isotopes at one or more of itsatoms. For example, the compounds may be radiolabeled with radioactiveisotopes, such as for example tritium (³H), deuterium (²H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C).

The cytotoxic dipeptide melphalan flufenamide clearly differs frommelphalan:

-   -   Difference in structure (melphalan flufenamide is an ethyl ester        at the C-terminal instead of the carboxylic acid in melphalan.        Melphalan is thereby a zwitterion, but melphalan flufenamide is        not).    -   Difference in size (melphalan flufenamide is a dipeptide, i.e.        approximately twice the size of melphalan).    -   Difference in lipophilicity, where melphalan flufenamide is        clearly more lipophilic.    -   Difference in stability in aqueous solutions. Melphalan is 10        000 times more stable in aqueous solutions compared to J1. J1 is        quickly hydrolyzed in water.    -   Difference in degradation pathways. The main degradation pathway        in melphalan flufenamide involves hydrolysis of the ethyl ester,        while the main degradation in melphalan relates to the        reactivity of the (chloro)alkyl groups.

Based on, but not limited to, the above differentiations, it is clearthat teachings on melphalan and, in particular preparations andformulations thereof, do not apply to melphalan flufenamide andpreparations and formulations thereof.

The inclusion of at least one excipient (such as Polysorbate 80 with itssurfactant properties) provides lyophilized preparation that is stableas such and water-soluble without the presence of an organic solvent ata sufficient rate compared to the degradation rate, and is therebyuseful in therapy and less toxic.

The lyophilized pharmaceutical preparation according to the inventionmay contain only melphalan flufenamide or a pharmaceutically acceptablesalt thereof, or a mixture of melphalan flufenamide with one or moredifferent cytotoxic dipeptides or pharmaceutically acceptable saltsthereof. Further, the lyophilized pharmaceutical preparation may containa mixture of two or more different pharmaceutically acceptable salts.

One aspect of the invention is a lyophilized pharmaceutical preparation,comprising

(i) melphalan flufenamide; andii) a combination of two or more excipients selected from the groupcomprising a polysorbate; a polyethylene glycol; β-cyclodextrin;α-cyclodextrin; hydroxypropyl-β-cyclodextrin;sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodiumsuccinate; propylene glycol; Cremophor EL; Dimethyl sulfoxide;D-mannitol; Trehalose; Sucrose; and an amino acid.

Yet an aspect of the invention is a lyophilized pharmaceuticalpreparation, comprising:

-   (i) melphalan flufenamide hydrochloride (J1); and-   (ii) a combination of two or more excipients selected from the group    comprising a polysorbate; a polyethylene glycol; β-cyclodextrin;    α-cyclodextrin; hydroxypropyl-β-cyclodextrin;    sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodium    succinate; propylene glycol; Cremophor EL; Dimethyl sulfoxide;    D-mannitol; Trehalose; Sucrose; and an amino acid.

In one embodiment of this aspect, said combination of excipients is amixture of Polysorbate 80 and PEG400.

Pharmaceutically acceptable salts for all aspects of the presentinvention may be, for instance, an acid-addition salt of a compounddescribed herein which is sufficiently basic, for example, anacid-addition salt with, for example, an inorganic or organic acid, forexample hydrochloric, hydrobromic, nitric, methansulphonic, sulphuric,phosphoric, trifluoroacetic, para-toluene sulphonic, 2-mesitylensulphonic, citric, acetic, tartaric, fumaric, lactic, succinic, malic,malonic, maleic, 1,2-ethanedisulphonic, adipic, aspartic,benzenesulphonic, benzoic, ethanesulphonic or nicotinic acid.

In this document, when the term “melphalan flufenamide” is used, it isalso intended to include pharmaceutically acceptable salt(s) thereof,even if this is not explicitly stated.

As mentioned hereinbefore, when melphalan flufenamide or apharmaceutically acceptable salt thereof is lyophilized in the presenceof a pharmaceutically acceptable excipient, such as any one selectedfrom a polysorbate; a polyethylene glycol; β-cyclodextrin;α-cyclodextrin; hydroxypropyl-β-cyclodextrin;sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodiumsuccinate; propylene glycol; Cremophor EL; Dimethyl sulfoxide;D-mannitol; Trehalose; Sucrose; and an amino acid; an unexpectedly highincrease in solubility of the lyophilized pharmaceutical preparation canbe obtained, which enables the direct dissolution of the lyophilizedmelphalan flufenamide in an aqueous solution, such as a physiologicallyacceptable solution. This is in contrast to a non-lyophilized melphalanflufenamide which is not possible to dissolve directly in an aqueoussolution but that first has to be dissolved in an organic solvent priorto dilution in an aqueous solution. It is therefore provided herein alyophilized pharmaceutical preparation comprising melphalan flufenamideor a pharmaceutically acceptable salt thereof, wherein melphalanflufenamide is lyophilized in the presence of an excipient. Preferably,said excipient is selected from polysorbate or polyethylene glycol, suchas Polysorbate 80 or PEG400.

Melphalan flufenamide or a pharmaceutically acceptable salt thereof maybe lyophilized in the presence of one or more of an excipient(s) (e.g.one, two, three, four, five, or more excipients). Examples of excipientsthat can be used as described herein include, without limitation,polysorbates such as Polysorbate 20, Polysorbate 40, Polysorbate 60, andPolysorbate 80; polyethylene glycols such as PEG 400 and PEG 300;β-cyclodextrin, α-cyclodextrin, sulfobutylether-β-cyclodextrin,hydroxypropyl-β-cyclodextrin, lactose, benzyl alcohol, disodiumsuccinate, propylene glycol, Cremophor EL, dimethyl sulfoxide,D-mannitol, trehalose, sucrose and amino acids such as histidine.

In one aspect of the invention, the excipient is selected from any oneof Polysorbate 80; PEG 400; β-cyclodextrin; α-cyclodextrin;hydroxypropyl-β-cyclodextrin; sulfobutylether-β-cyclodextrin; lactose;benzyl alcohol; disodium succinate; propylene glycol; PEG 300; CremophorEL; Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose; and histidine.

In one aspect of the invention, the excipient is selected fromPolysorbate 80; PEG 400; lactose; benzyl alcohol; disodium succinate;propylene glycol; PEG 300; Cremophor EL; Dimethyl sulfoxide; D-mannitol;Trehalose; Sucrose; and histidine, or a combination of two or more ofsaid excipients.

In one embodiment of this aspect, the excipient is selected fromPolysorbate 80 and PEG 400, or a combination of said two excipients.

The amount of excipient such as Polysorbate 80, PEG 400 orβ-cyclodextrin, is typically about 10-100% by weight of the amount ofmelphalan flufenamide, such as 100, 90, 80, 70, 60, 50, 40, 30, 20 or10% by weight of the amount of melphalan flufenamide.

In yet an aspect of the invention, the amount of the excipient, such asPolysorbate 80, PEG 400 or β-cyclodextrin, is typically about 10-50% byweight of the amount of melphalan flufenamide, such as 100, 90, 80, 70,60, 50, 40, 30, 20 or 10% by weight of the amount of melphalanflufenamide.

In one embodiment of this aspect, the excipient represents Polysorbate80 or PEG 400, and the amount thereof is typically about 10-50% byweight of the amount of melphalan flufenamide, such as 100, 90, 80, 70,60, 50, 40, 30, 20 or 10% by weight of the amount of melphalanflufenamide.

Still an aspect of the invention is a lyophilized pharmaceuticalpreparation comprising

(i) melphalan flufenamide, or a pharmaceutically acceptable saltthereof; and(ii) at least one excipient selected from the group comprising

-   -   Polysorbate 80; PEG 400; β-cyclodextrin; α-cyclodextrin;        hydroxypropyl-β-cyclodextrin; sulfobutylether-β-cyclodextrin;        lactose; benzyl alcohol; disodium succinate; propylene glycol;        PEG 300; Cremophor EL; Dimethyl sulfoxide; D-mannitol;        Trehalose; Sucrose; and histidine;    -   wherein the amount of the excipient is about 10-100% by weight        of melphalan flufenamide.

In one embodiment of this aspect, the at least one excipient selectedfrom Polysorbate 80 and PEG 400.

In another embodiment of this aspect, melphalan flufenamide isrepresented by melphalan flufenamide hydrochloride (J1).

Still an aspect of the invention is a lyophilized pharmaceuticalpreparation comprising

(i) melphalan flufenamide, or a pharmaceutically acceptable saltthereof; and(ii) at least one excipient selected from the group comprising

-   -   Polysorbate 80; PEG 400; β-cyclodextrin; α-cyclodextrin;        hydroxypropyl-β-cyclodextrin; sulfobutylether-β-cyclodextrin;        lactose; benzyl alcohol; disodium succinate; propylene glycol;        PEG 300; Cremophor EL; Dimethyl sulfoxide; D-mannitol;        Trehalose; Sucrose; and histidine;    -   wherein the amount of the excipient is about 10-50% by weight of        melphalan flufenamide hydrochloride (J1).

In one embodiment of this aspect, the at least one excipient selectedfrom Polysorbate 80 and PEG 400.

In another embodiment of this aspect, melphalan flufenamide isrepresented by melphalan flufenamide hydrochloride (J1).

Yet an aspect of the invention is a lyophilized pharmaceuticalpreparation comprising

(i) melphalan flufenamide hydrochloride (J1); and(ii) at least one excipient selected from the group comprising

-   -   Polysorbate 80; PEG 400; β-cyclodextrin; α-cyclodextrin;        hydroxypropyl-β-cyclodextrin; sulfobutylether-β-cyclodextrin;        lactose; benzyl alcohol; disodium succinate; propylene glycol;        PEG 300; Cremophor EL; Dimethyl sulfoxide; D-mannitol;        Trehalose; Sucrose; and histidine;    -   wherein the amount of the excipient is about 10-100% by weight        of melphalan flufenamide hydrochloride (J1).

In one embodiment of this aspect, the at least one excipient selectedfrom Polysorbate 80 and PEG 400.

Yet an aspect of the invention is a lyophilized pharmaceuticalpreparation comprising

-   (i) melphalan flufenamide hydrochloride (J1); and-   (ii) at least one excipient selected from the group comprising    Polysorbate 80; PEG 400; β-cyclodextrin; α-cyclodextrin;    hydroxypropyl-β-cyclodextrin; sulfobutylether-β-cyclodextrin;    lactose; benzyl alcohol; disodium succinate; propylene glycol; PEG    300; Cremophor EL; Dimethyl sulfoxide; D-mannitol; Trehalose;    Sucrose; and histidine;    -   wherein the amount of the excipient is about 10-50% by weight of        melphalan flufenamide hydrochloride (J1).

In one embodiment of this aspect, the at least one excipient selectedfrom Polysorbate 80 and PEG 400.

In one embodiment of the invention, the amount of excipient, such asPolysorbate 80 or PEG 400, may be up to the clinically acceptableamount.

In one embodiment of the invention, the amount of excipient, such asPolysorbate 80 or PEG 400, may be up to the clinically acceptableamount.

When used as the only excipient, the amount of Polysorbate 80 or PEG400, is e.g. about 50% by weight of the amount of melphalan flufenamidehydrochloride (J1).

An aspect of the invention is a combination of the excipientsPolysorbate 80 and PEG 400.

An aspect of the invention is a combination of the excipientsPolysorbate 80, PEG 400 and β-cyclodextrin, such as 80% by weight ofPolysorbate 80, 80% by weight of PEG 400 and 50% by weight ofβ-cyclodextrin, of the amount of melphalan flufenamide. A lyophilizedpharmaceutical preparation of a melphalan derivative or apharmaceutically acceptable salt thereof, may in accordance with theinvention comprise one or more melphalan derivative(s) or apharmaceutically acceptable salt(s) thereof, and one or moreexcipient(s) as defined herein.

As mentioned hereinbefore, one effect of the presence of an excipientduring the lyophilization is that the resulting lyophilizedpharmaceutical preparation comprising melphalan flufenamide, has anenhanced solubility in aqueous solutions, such as a physiologicallyacceptable solution, compared to when melphalan flufenamide islyophilized without an excipient as described herein. In particular, thesolubility in aqueous solutions of melphalan flufenamide whenlyophilized in the presence of an excipient(s) is higher compared to thesolubility of the non-lyophilized product. This increased solubility ofmelphalan flufenamide, in particular when lyophilized in the presence ofan excipient as herein described, compared to the non-lyophilizedproduct, has substantial advantages when it comes to administration ofmelphalan flufenamide to a patient.

Due to a low solubility of non-lyophilized melphalan flufenamide inaqueous physiologically acceptable solutions used for administration ofthe drug to a patient, it is necessary to first dissolve thenon-lyophilized melphalan flufenamide in an organic solvent, such asDMA. Melphalan flufenamide is therefore often stored dissolved in DMA.It has previously not been possible to directly dissolve the melphalanflufenamide in an aqueous solution, but organic solvents have had to beused. Once dissolved in the organic solvent, this solution of melphalanflufenamide and organic solvent can be dissolved in physiologicallyacceptable solutions for administration to a subject.

As melphalan flufenamide is very toxic, in order to minimize theexposure of medical personnel to such drugs, special devices fortransferring the drugs after dissolution in organic solvents to thesolution for administration, are used. These transfer devices are oftenplastic tubings comprising polycarbonate. However, such tubings aresensitive to and may be destroyed by organic solvents, such as DMA.Therefore, in the cases where the drug to be administered is dissolvedin such an organic solvent, it may not be possible to use the transferdevice, and the dissolved drug instead has to be directly added to thephysiologically acceptable solution used for administration just beforethe time of administration to the patient. This can be hazardous for themedical staff, who then are at risk being exposed to the toxic drug.

As mentioned above, lyophilization of melphalan flufenamide increasesits solubility in physiologically acceptable solutions. This increasecan be even more pronounced when melphalan flufenamide is lyophilized inthe presence of one or more excipients. As described herein, whenmelphalan flufenamide is lyophilized in the presence of an excipient asdisclosed herein, the solubility of melphalan flufenamide can beincreased, in comparison to the non-lyophilized melphalan flufenamide.The use of an organic solvent, such as DMA, to first dissolve melphalanflufenamide can be avoided.

Melphalan flufenamide which has been lyophilized in the presence of atleast one excipient, such as a polysorbate which for example may bePolysorbate 80; a polyethylene glycol which for example may be PEG 400or PEG 300; β-cyclodextrin; α-cyclodextrin;hydroxypropyl-β-cyclodextrin; sulfobutylether-β-cyclodextrin; lactose;benzyl alcohol; disodium succinate; propylene glycol; Cremophor EL;Dimethyl sulfoxide; D-mannitol; Trehalose; Sucrose; or an amino acidsuch as histidine; or a combination of two or more of these excipients;can be directly dissolved in a physiologically acceptable solution, suchas about 4.5-5.5 wt %, e.g. about 5%, glucose solution or an aqueousNaCl solution (e.g. about 0.9 wt % NaCl). Thereby, devices comprisingpolycarbonate and which are used for the administration of melphalanflufenamide are possible to use, minimizing the risk for exposing themedical personnel to the drug. Also, in this way administering the toxicDMA to the patient is avoided. This allows for directly preparing thesolution comprising melphalan flufenamide at a concentration suitablefor administration to the patient. Alternatively, a concentratedsolution comprising a lyophilized pharmaceutical preparation ofmelphalan flufenamide in a physiologically acceptable solution may firstbe prepared and then transferred to the bag for infusion using thecommonly used transfer devices.

Also, when melphalan flufenamide is dissolved in DMA, an adduct betweenthe melphalan flufenamide and the DMA can be formed. By using alyophilized pharmaceutical preparation provided in accordance with theinvention, it is possible to dissolve the lyophilized melphalanflufenamide directly in a physiologically acceptable solution, avoidingfirst dissolving the melphalan flufenamide in DMA. Thereby, theformation of a DMA-melphalan flufenamide adducts can be avoided andneither the adduct nor the DMA have to be administered to the patient.

There is also provided a pharmaceutical composition comprising alyophilized pharmaceutical preparation of melphalan flufenamide orpharmaceutically acceptable salt thereof as defined herein, optionallyobtainable by the method for preparing such a lyophilized preparationdisclosed herein. Such a pharmaceutical composition may further comprisea physiologically acceptable solution, such as an aqueous NaCl (e.g.about 0.9 wt %) or glucose solution (e.g. about 4.5-5.5 wt %, such asabout 5 wt %, glucose). This pharmaceutical composition may be aconcentrated solution intended for dilution before administration to asubject or as a solution enabling direct administration to a patient.

Due to the increased solubility of melphalan flufenamide afterlyophilization in the presence of one or more excipients as describedherein, it is possible to prepare a dissolved melphalan flufenamidesolution, such as a pharmaceutical composition comprising a melphalanflufenamide or pharmaceutically acceptable salt thereof, which issubstantially free from organic solvents such as DMA, dichloromethane,tetrahydrofuran, 2-methyl tetrahydrofuran, ethyl acetate, acetone,dimethylformamide, acetonitrile, dimethyl sulfoxide, dioxane, diethylether, acetic acid, n-butanol, isopropanol, n-propanol, tert-butanol,sec-butanol, methanol, ethanol, and acetic acid. By “substantially free”is in this document meant that the pharmaceutical composition comprisesonly trace amounts of an organic solvent, such as less than about atotal of about 0.1 wt % of an organic solvent. In one aspect, thelyophilized preparation or the pharmaceutical composition does notcontain any measurable amounts of an organic solvent. Such preparationswould be less toxic and therefore more tolerated by a patient, ie givingless side effect such as vomiting, nausea or other general symptoms wheninfused.

In one aspect of the invention, there is provided a lyophilizedpharmaceutical preparation as described herein, which is free, orsubstantially free from organic solvents.

The pharmaceutical composition may consist of a lyophilizedpharmaceutical preparation as disclosed herein, comprising melphalanflufenamide or pharmaceutical salt thereof, and the physiologicallyacceptable solution, such as a glucose solution. As disclosedhereinbefore, the melphalan derivative may be melphalan flufenamide or amixture of melphalan flufenamide and one or more different cytotoxicdipeptides, either lyophilized together or separately.

The pharmaceutical composition may be obtainable by dissolving melphalanflufenamide or a pharmaceutical salt thereof in a physiologicallyacceptable solution. A method for preparing a pharmaceutical compositioncomprising the step of dissolving the lyophilized pharmaceuticalpreparation comprising melphalan flufenamide or a pharmaceuticallyacceptable salt thereof in a physiologically acceptable solution istherefore also provided herein.

The wording a “physiologically acceptable solution” is herein defined,is an aqueous solution, such as a NaCl solution (such as about 0.9 wt-%NaCl) or glucose solution, such as about 4.5-5.5 wt-% glucose, e.g.about 5 wt-%, or another physiologically acceptable solution. Any suchsolution may optionally be buffered.

A pharmaceutical composition comprising lyophilized melphalanflufenamide and a physiologically acceptable solution for directadministration to a subject, generally comprises melphalan flufenamideat a concentration of about 1 mg/ml or less, such as about 0.2 mg/ml.However, the pharmaceutical composition may comprise melphalanflufenamide in a concentration of up to about 4 mg/ml for dilution in aphysiologically acceptable solution before administration to a patient.

One aspect of the invention provides a method for preparing alyophilized pharmaceutical preparation, whereby:

-   -   a) melphalan flufenamide, or a pharmaceutically acceptable salt        thereof, is dissolved in an organic solvent to obtain a        melphalan flufenamide solution;    -   b) water is added to the melphalan flufenamide solution in order        to obtain an aqueous melphalan flufenamide solution, in a        concentration of about 0.2-3.0 mg/ml;    -   c) at least one excipient selected from the group comprising a        polysorbate; a polyethylene glycol; β-cyclodextrin;        α-cyclodextrin; hydroxypropyl-β-cyclodextrin;        sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol;        disodium succinate; propylene glycol; Cremophor EL; Dimethyl        sulfoxide; D-mannitol; Trehalose; Sucrose and an amino acid is        added to the melphalan flufenamide solution; and    -   d) the aqueous melphalan flufenamide solution containing        excipient(s) is subjected to lyophilization.

In one embodiment of this aspect, there is provided a method, whereby:

-   -   a) melphalan flufenamide, or a pharmaceutically acceptable salt        thereof, is dissolved in an organic solvent;    -   b) water is added to the solution obtained in step a) in order        to obtain a solution of said melphalan flufenamide or a        pharmaceutically acceptable salt thereof, in a concentration of        about 0.2-3.0 mg/ml;    -   c) at least one excipient selected from the group comprising a        polysorbate; a polyethylene glycol; β-cyclodextrin;        α-cyclodextrin; hydroxypropyl-β-cyclodextrin;        sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol;        disodium succinate; propylene glycol; Cremophor EL; Dimethyl        sulfoxide; D-mannitol; Trehalose; Sucrose and an amino acid is        added to the solution obtained in step b); and    -   d) the solution obtained in step c) is subjected to        lyophilization.

The organic solvent may be selected from any one of ethanol, ethanolcontaining acid, glycerin, propylene glycol, benzyl alcohol,dimethylacetamide (DMA), N-methyl-2-pyrrolidone, isopropanol, n-butanol,tert-butanol, methyl tert-butyl ether, propylene glycol,dimethylsulfoxide, tetrahydrofuran, 2-methyl tetrahydrofuran, acetone,dimethylformamide, acetonitrile, dioxane, acetic acid, lactic acid,propionic acid, n-butanol, isopropanol, n-propanol, tert-butanol,sec-butanol, methanol, and a mixture of ethanol and water. Preferably,said organic solvent is ethanol.

The excipient may be selected from the group comprising Polysorbate 80;PEG 400; β-cyclodextrin; α-cyclodextrin; hydroxypropyl-β-cyclodextrin;sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodiumsuccinate; propylene glycol; PEG 300; Cremophor EL; Dimethyl sulfoxide;D-mannitol; Trehalose; Sucrose; and histidine. Preferably, saidexcipient is selected from Polysorbate 80 and PEG 400.

The melphalan flufenamide in said methods is preferably melphalanflufenamide hydrochloride (J1).

An aspect of the present invention is a method for the preparation of alyophilized pharmaceutical preparation as herein described, whereby

-   -   a) melphalan flufenamide, or a pharmaceutically acceptable salt        thereof, is dissolved in an organic solvent;    -   b) water is added to the solution obtained in step a) in order        to obtain a solution of said melphalan flufenamide or a        pharmaceutically acceptable salt thereof, in a concentration of        about 0.2-3.0 mg/ml;    -   c) at least one excipient as herein defined, is added to the        solution obtained in step b); and    -   d) the solution obtained in step c) is subjected to        lyophilization.

Preferably, said organic solvent is ethanol.

An aspect of the present invention is a method for the preparation of alyophilized pharmaceutical preparation as herein described, whereby

-   -   a) melphalan flufenamide hydrochloride (J1), is dissolved in an        organic solvent;    -   b) water is added to the solution obtained in step a) in order        to obtain a solution of said melphalan flufenamide hydrochloride        (J1), or a pharmaceutically acceptable salt thereof, in a        concentration of about 0.2-3.0 mg/ml;    -   c) at least one excipient as herein defined, is added to the        solution obtained in step b); and    -   d) the solution obtained in step c) is subjected to        lyophilization.

Examples of organic solvents useful for dissolving melphalanflufenamide, or a pharmaceutically acceptable salt thereof in step a),may be any one selected from ethanol, ethanol containing acid, glycerin,propylene glycol, benzyl alcohol, dimethylacetamide (DMA),N-methyl-2-pyrrolidone, isopropanol, n-butanol, tert-butanol, methyltert-butyl ether, propylene glycol, dimethylsulfoxide, tetrahydrofuran,2-methyl tetrahydrofuran, acetone, dimethylformamide, acetonitrile,dioxane, acetic acid, lactic acid, propionic acid, n-butanol,isopropanol, n-propanol, tert-butanol, sec-butanol, methanol, and amixture of ethanol and water.

An aspect of the present invention is a method for the preparation of alyophilized pharmaceutical preparation as herein described, whereby

-   a) melphalan flufenamide, or a pharmaceutically acceptable salt    thereof, is dissolved in an organic solvent selected from any one of    ethanol, ethanol containing acid, glycerin, propylene glycol, benzyl    alcohol, dimethylacetamide (DMA), N-methyl-2-pyrrolidone,    isopropanol, n-butanol, tert-butanol, methyl tert-butyl ether,    propylene glycol, dimethylsulfoxide, tetrahydrofuran, 2-methyl    tetrahydrofuran, acetone, dimethylformamide, acetonitrile, dioxane,    acetic acid, lactic acid, propionic acid, n-butanol, isopropanol,    n-propanol, tert-butanol, sec-butanol, methanol, and a mixture of    ethanol and water;-   b) water is added to the solution obtained in step a) in order to    obtain a solution of said melphalan flufenamide or a    pharmaceutically acceptable salt thereof, in a concentration of    about 0.2-3.0 mg/ml;-   c) at least one excipient as herein defined, is added to the    solution obtained in step b); and-   d) the solution obtained in step c) is subjected to lyophilization.

An aspect of the present invention is is a method for the preparation ofa lyophilized pharmaceutical preparation as herein described, whereby

-   a) melphalan flufenamide hydrochloride (J1), is dissolved in an    organic solvent selected from any one of ethanol, ethanol containing    acid, glycerin, propylene glycol, benzyl alcohol, dimethylacetamide    (DMA), N-methyl-2-pyrrolidone, isopropanol, n-butanol, tert-butanol,    methyl tert-butyl ether, propylene glycol, dimethylsulfoxide,    tetrahydrofuran, 2-methyl tetrahydrofuran, acetone,    dimethylformamide, acetonitrile, dioxane, acetic acid, lactic acid,    propionic acid, n-butanol, isopropanol, n-propanol, tert-butanol,    sec-butanol, methanol, and a mixture of ethanol and water;-   b) water is added to the solution obtained in step a) in order to    obtain a solution of said melphalan flufenamide hydrochloride (J1),    or a pharmaceutically acceptable salt thereof, in a concentration of    about 0.2-3.0 mg/ml;-   c) at least one excipient as herein defined, is added to the    solution obtained in step b); and-   d) the solution obtained in step c) is subjected to lyophilization.

An aspect of the present invention is a method for the preparation of alyophilized pharmaceutical preparation as herein described, whereby

-   a) melphalan flufenamide hydrochloride (J1), is dissolved in an    organic solvent;-   b) water is added to the solution obtained in step a) in order to    obtain a solution of said melphalan flufenamide hydrochloride (J1),    in a concentration of about 0.2-3.0 mg/ml;-   c) at least one excipient as herein defined, is added to the    solution obtained in step b); and-   d) the solution obtained in step c) is subjected to lyophilization;    wherein said at least one excipient is selected from Polysorbate 80    and PEG400.

When ethanol containing acid is used for dissolving melphalanflufenamide or a pharmaceutically acceptable salt thereof in step a) inthe method above, the acid can be HCl, in a concentration of for example5-20 mM, or the HCl concentration may for example be 10 mM, in theethanol.

When melphalan flufenamide or a pharmaceutically acceptable salt thereofis dissolved in ethanol and water, the concentration of ethanol may beabout 10-100 vol-%, such as 10-90 vol-%, 50-90 vol-%, or about 70 vol-%.

The water used for dissolving and/or diluting samples of a lyophilizedpharmaceutical preparation in accordance with the present invention, issterile or purified water, or water for injection (WFI).

When ethanol is used for dissolving melphalan flufenamide orpharmaceutically acceptable salt thereof, the solution obtained in stepa) is diluted in step b) so that the concentration of ethanol, is about2%-100% by volume, such as about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80,90 or 100%, or such as 5-15%, or such as 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15%. Typically, the concentration of ethanol after the dilutionstep b) is about 9%.

The solution obtained in step b) may be sterile filtered before thelyophilization step c).

The lyophilization step c) comprises the typical freezing and primaryand secondary drying steps as described herein. Information about howlyophilization is performed may be found e.g. in Rey, L. and May, J.Freeze Drying/Lyophilization of Pharmaceutical and Biological Products(2010), ISBN 978-1439B2575-4. In the freezing step, the sample is forexample frozen in a bath of dry ice-acetone at a temperature of about−70° C. to −90° C., such as about −70° C., −75° C., −78° C., −80° C.,−82° C., −85° C., −88° C. or −90° C. for example for 10 minutes to 120minutes.

Alternatively, the sample may be frozen in a freezer at a temperatureabout −14° C. to −25° C., such as −14° C., −16° C., −18° C., −20° C.,−22° C., or −25° C., for example for about 10 min to 24 hours. It isalso possible to freeze the sample in liquid nitrogen.

Step c) may be performed by applying conventional techniques forlyophilization, see e.g. Rey, L. and May, J. FreezeDrying/Lyophilization of Pharmaceutical and Biological Products (2010),ISBN 978-1439B2575-4.

For example, in the primary drying step, the pressure can be lowered toabout to about 0.1 mbar to 50 mbar, such as 1 mbar to 10 mbar. Thetemperature is typically below 0° C., such as −50 to 0° C., or −20 to−1° C., e.g. −50, −40, −30, −20, −10, or −5° C. This phase may forexample last for 4 hours to 48 hours, e.g. 12 hours to 24 hours.

In the final secondary drying step, when most of the water hasevaporated, the temperature may be as in the primary drying step orabove 0° C.

When one or more excipients as defined herein are to be present duringthe lyophilization, these can be added in step b) prior to or afterdiluting the solution obtained in step a) and prior to performing thelyophilization. The excipients may be added in powder form but aregenerally added as an aqueous solution. The excipients can therefore bepresent during the lyophilization.

The present invention is also directed to a lyophilized pharmaceuticalpreparation as defined herein obtainable by the above disclosed method.

It is also provided herein a kit of parts comprising:

-   (i) a first container comprising a lyophilized pharmaceutical    preparation comprising melphalan flufenamide as described herein;    and-   (ii) a second container comprising a physiologically acceptable    solution, such as a NaCl solution (such as about 0.9 wt % NaCl) or a    glucose solution, such as about 4.5-5.5 wt % glucose solution, e.g.    about 5 wt % glucose solution, or other physiologically acceptable    solution.

Such a kit may also comprise a device for mixing the contents of the twocontainers with each other and/or for transferring the resulting mixtureto a device, such as a bag comprising a glucose solution, for theadministration to a patient.

Such a kit may consist of the first container comprising a lyophilizedpharmaceutical preparation comprising melphalan flufenamide as describedherein and the second container comprising the physiologicallyacceptable solution. Melphalan flufenamide in the kit may also be inadmixture with a pharmaceutically acceptable carrier and/or excipient.One example is 5% glucose with e.g. 1% albumin or another protein orcompound. The amount of physiologically acceptable solution may eitherbe a small amount in order to prepare a concentrated solution of thelyophilized pharmaceutical preparation comprising melphalan flufenamide,or a larger amount in order to enable the preparation of a solutionhaving the desired concentration for administration to a patient.Alternatively, the kit may comprise both a container comprising aphysiologically acceptable solution for preparing a concentratedsolution of the infusion, comprising a larger amount of aphysiologically acceptable solution for preparation of the more dilutedsolution for administration to a subject.

A lyophilized pharmaceutical preparation, pharmaceutical composition orkit provided herein may comprise only melphalan flufenamide or apharmaceutically acceptable salt thereof as an antitumoral agent.However, melphalan flufenamide may also be combined with one or moreantitumoral agents, such as other antitumoral substances such asgemcitabine, etoposide, doxorubicine or taxanes or other therapeuticallyeffective substances. When combined with other antitumoral agents thesemay either be mixed with melphalan flufenamide or pharmaceuticallyacceptable salt thereof before lyophilisation and consequentlylyophilized together with melphalan flufenamide or pharmaceuticallyacceptable salt thereof or combined with the lyophilized melphalanflufenamide or pharmaceutically acceptable salt thereof afterlyophilisation, such as in a kit or a pharmaceutical composition.Lyophilized melphalan flufenamide may also be mixed with one or moreantitumoral substances in dry form, even though not lyophilized, afterlyophilisation of melphalan flufenamide or pharmaceutically acceptablesalt thereof.

Melphalan flufenamide provided herein have a cytotoxic activity and maytherefore be used in the prevention and/or treatment of cancer asdescribed elsewhere (see e.g. WO 01/96367). A reduction of tumor cellsurvival of these compounds was in WO 01/96367 demonstrated fordifferent hematological and/or solid tumors, e.g. lung cancer, myeloma,lymphoma, leukemia, breast cancer, and ovarian carcinoma. Further, thesecompounds were in WO 01/96367 demonstrated to circumvent melphalanresistance. These compounds may therefore be used in the preventionand/or treatment of cancer, reducing tumor growth and/or killing tumorcells. Thus, the compounds may be used for curing and/or prolonging thesurvival of patients afflicted with cancer diseases.

Also provided herein is the lyophilized pharmaceutical preparation, kitor pharmaceutical composition as disclosed and claimed herein, for useas a medicament. The invention is also directed to such a lyophilizedpharmaceutical preparation, kit or pharmaceutical composition, for usein the treatment and/or prevention of cancer, such as ovarian cancer,lung cancer, bladder cancer, mesothelioma, multiple myeloma, breastcancer and/or any other solid or hematological cancer.

An aspect of the present invention is the use of a lyophilizedpharmaceutical preparation, kit or pharmaceutical composition asdisclosed and claimed herein, for the preparation of a medicament forthe treatment and/or prevention of cancer, such as ovarian cancer, lungcancer, bladder cancer, mesothelioma, multiple myeloma, breast cancerand/or any other solid or hematological cancer.

Yet an aspect of the present invention provides a lyophilizedpharmaceutical preparation, kit or pharmaceutical composition comprisingmelphalan flufenamide hydrochloride (J1) in combination with anotherdrug useful in the treatment of cancer, for use in treatment and/orprevention of cancer, such as ovarian cancer, lung cancer, bladdercancer, mesothelioma, multiple myeloma, breast cancer and/or any othersolid or hematological cancer.

Yet an aspect of the present invention is a method for the treatment ofand/or prevention of cancer, such as ovarian cancer, lung cancer,bladder cancer, mesothelioma, multiple myeloma, breast cancer and/or anyother solid or hematological cancer. The method can comprise theadministration of a lyophilized pharmaceutical preparation, a kit or apharmaceutical composition as provided herein in a therapeuticallyeffective dose to a subject in need thereof. The subject is typically ahuman or a domestic animal.

Yet an aspect of the present invention is a method for the treatment ofand/or prevention of cancer, such as ovarian cancer, lung cancer,bladder cancer, mesothelioma, multiple myeloma, breast cancer and/or anyother solid or hematological cancer, wherein the lyophilizedpharmaceutical preparation, a kit or a pharmaceutical compositioncomprising melphalan flufenamide hydrochloride (J1) is provided in atherapeutically effective dose to a subject in need thereof, incombination with another drug, useful in the treatment of cancer. Thesubject is typically a human or a domestic animal.

The administration of a lyophilized pharmaceutical preparation, a kit ora pharmaceutical composition to a subject in need thereof may take placeby intravenous injections. It is also possible to administer lyophilizedmelphalan flufenamide or a pharmaceutical composition comprising suchlyophilized melphalan flufenamide in body cavities, such as instillationin the bladder, or in peritoneal or pleural cavities.

Melphalan flufenamide or a pharmaceutically acceptable salt thereof maybe administered in an amount of about 20-130 mg, such as 30-75 mg, forexample 50 mg total amount of melphalan flufenamide per administration.The pharmaceutical composition or kit provided herein comprisingmelphalan flufenamide may therefore have an amount of lyophilizedmelphalan flufenamide such that this amount can be administered.

Lyophilized melphalan flufenamide or a pharmaceutically acceptable saltthereof may be administered daily, every second or third day, weekly,every second, third or 4^(th) week or even as a high single dose (e.g.before transplantation) depending on the subject and cancer form to betreated.

The wording “prevention” as used herein, is intended to include therapyin a patient that has been subjected to chemotherapy against any cancerform as herein described, and who is subjected to continued therapy withthe aim of preventing any methastasis occurring from said cancer.

Yet an aspect of the present invention provides use of an excipientselected from the group comprising Polysorbate 80; PEG 400;β-cyclodextrin; α-cyclodextrin; hydroxypropyl-β-cyclodextrin;sulfobutylether-β-cyclodextrin; lactose; benzyl alcohol; disodiumsuccinate; propylene glycol; PEG 300; Cremophor EL; Dimethyl sulfoxide;D-mannitol; Trehalose; Sucrose; and histidine, in a lyophilizedpreparation of melphalan flufenamide, or a pharmaceutically acceptablesalt thereof, for decreasing the reconstitution time of the lyophilizedpreparation of melphalan flufenamide, or a pharmaceutically acceptablesalt thereof, when reconstituted in an aqueous solvent.

Said melphalan flufenamide, or a pharmaceutically acceptable saltthereof, is preferably melphalan flufenamide hydrochloride (J1).

Said excipient is preferably selected from Polysorbate 80 and PEG 400.

Said melphalan flufenamide, or a pharmaceutically acceptable saltthereof, is preferably dissolved in ethanol prior to subjecting saidmelphalan flufenamide to said excipient.

In this document “lyophilization”, “freeze-drying”, “lyophilized”,“freeze-dried”, and the like may be used interchangeably.

Polysorbate 80 (having the chemical name Polyoxyethylene 20 sorbitanmonooleate and the CAS registry number 9005-65-6) is commerciallyavailable from e.g. Fluka or Sigma-Aldrich.

PEG 400 has the empirical formula HOCH₂(CH₂OCH₂)_(m)CH₂OH, where m is8.7, and the average molecular weight is 380-420, and is commerciallyavailable from e.g. Fluka or Sigma-Aldrich.

PEG 300 has the empirical formula HOCH₂(CH₂OCH₂)_(m)CH₂OH, where m is6.4, and the average molecular weight is 285-315, and is commerciallyavailable from e.g. Fluka or Sigma-Aldrich.

Cremophor EL® is a trade mark sold by Sigma-Aldrich, and isPolyoxyethylene castor oil having the CAS Registry Number 61791-12-6.

Exemplary cytotoxic dipeptides that can be used as described herein arealso disclosed in WO01/96367 and can have the formula V

whereinR₁ is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂, alkylamino,cycloalkylamino or arylamino;R₃ is NH₂, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl, N(CH₂CH₂Cl)₂, NO₂, F,CF₃ or H; andR₄ is a natural or modified cyclic or aromatic amino acid, or H; as wellas pharmaceutically acceptable salts thereof.

Also, cytotoxic peptides that can be used as described herein includepeptides of the formula I or V, wherein R₃ is F. Dipeptides are examplesof peptides of the formula I or V, wherein R₁ is alkyloxy; R₃ is F, CF₃,H, OH, O-alkyl, NO₂, N(CH₂CH₂Cl)₂, NH-acyl or NH₂; and R₄ is H.

Tripeptides are example of peptides of the formula I or V, wherein R₁ isalkyloxy; R₃ is F, CF₃, H, OH, O-alkyl, NH-acyl, NO₂, N(CH₂CH₂Cl)₂ orNH₂; and R₄ is a natural or modified cyclic or aromatic amino acid.

Melphalan flufenamide, or a pharmaceutically acceptable salt thereof,may be prepared as disclosed in WO 01/96367, which disclosure isincorporated by reference. Example 1 of WO 01/96367 discloses asynthetic procedure for making melphalan flufenamide(L-melphalanyl-L-p-fluorophenylalanine ethyl ester), as well as itshydrochloride salt-melphalan flufenamide hydrochloride J1(L-melphalanyl-L-p-fluorophenylalanine ethyl ester, compound J1), whichdisclosure is incorporated herein.

The dipeptide derivatives disclosed in WO01/96367 can be synthesisedfrom tert-butoxycarbonyl(Boc)-protected melphalan as disclosed thereinand can be lyophilized and used as described herein. Also, WO01/96367discloses the preparation of tripeptide derivatives, in whichBoc-protected amino acids were coupled to the melphalan containingdipeptide derivative using EDC/NMM/HOBt as coupling reagents (EDC istriethylamine or 1-[3-dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride, NMM is N-methylmorpholine and HOBt is1-hydroxybenzotriazole). Such tripeptide derivatives may be lyophilizedand used as described herein.

Examples of derivatives of melphalan that can be lyophilized and used asdescribed herein in all aspects include, without limitation, melphalanflufenamide, L-melphalanyl-L-p-fluorophenylalanine isopropyl ester(JV28), L-prolinyl-L-melphalanyl-L-p-fluorophenylalanine ethyl ester(J3) (FIG. 7) and pharmaceutically acceptable salts thereof. Thesecompounds are disclosed previously in WO01/96367, which also providesmethods for their preparation. Melphalan flufenamide, JV28 and J3 may betransformed into melphalan in the body. In WO 01/96367, thesederivatives were demonstrated to have an increased cell killing activityagainst tumors, even when used at lower concentrations than melphalan.In addition, melphalan resistance could be circumvented.

The invention will be further described by way of the followingexamples, which do not limit the scope of the invention.

EXPERIMENTAL SECTION Example 1 Lyophilization of Melphalan FlufenamideHydrochloride (J1) Under Different Conditions

It this experiment lyophilization of melphalan flufenamide hydrochloride(J1), under various conditions was tested.

Example 1A

Weighed amounts of J1 were dissolved in various volumes of deionizedwater in ultrasound bath with slight heating to get clear solutions. Thesamples were frozen in a bath of dry ice-acetone (−78° C., samplesA1-A3) or in a freezer at −16° C. (samples B1-B3). Freeze-drying wasthen conducted for 16 h at a pressure of 1 mbar at room temperature witha dry ice-acetone (−78° C.) trap between the drying flask and the pump.

The visual appearance after drying was as summarized in Table 1.

TABLE 1 Six different solutions of J1 at various freeze concentrationsor temperatures. Exp no mg J1 mL water conc (mg/mL) appearance afterdrying J1A1 2.4 6 0.4 white fluffy J1A2 2.7 27 0.1 white fluffy - someincompletely dry J1A3 2.5 10 0.25 white fluffy J1B1 2.7 6.75 0.4 whitesolid J1B2 2.5 25 0.1 light yellow powder J1B3 2.8 11.2 0.25 whitefluffy

Example 1B

Samples of the dried compounds were dissolved in 50% aqueousacetonitrile and analyzed by HPLC (ACE-column, C8, 50×3 mm, 10-97% CH₃CNin 3 min, 1 mL/min). In one case (J1A1) the aqueous solution wasanalyzed by HPLC before freeze-drying (J1A1-start). The purities afterdrying were as summarized in Table 2.

TABLE 2 Purity after freeze drying. Rt = Retention time Exp no J1:Rt2.27 (%) Rt 1.87 (%) Rt 1.44 (%) J1A1-start 88 12 J1A1 79 21 J1A2 80 20J1A3 41 45 14 J1B1 34 42 25 J1B2 36 43 21 J1B3 79 21

Example 1

Next it was tested to use either slightly acidic water (for example0.01% HCl) to enhance the speed of dissolution or to first dissolve J1in ethanol, before adding water (neutral or slight acidic).

Three samples of J1 were prepared by dissolving melphalan flufenamide(ca 3 mg) in 70% aq. ethanol (0.5 mL). The solutions were diluted with 5mM HCl to give a concentration of 0.4 mg/mL. Since melphalan flufenamidedissolved rapidly in aq. ethanol it was not necessary to use ultrasoundbath or heating to get a clear solution. The solutions were then frozenin a bath of dry ice-acetone (−78° C.) trap between the drying flask andthe pump. The visual appearance after drying was as summarized in Table3.

TABLE 3 Three replicates of J1 dissolved in ethanol and acid. Exp no mgJ1 mL HCl conc (mg/mL) appearance after drying J1C1 3.0 7.0 0.4 whitesolid some of which adhered to the glass J1C2 3.2 7. 0.4 white solidsome of which adhered to the glass J1C3 2.9 6.75 0.4 white solid some ofwhich adhered to the glass

Example 1D

Two HPLC runs were done on each sample: one from the solid compound thatcould be removed from the flask and one by dissolving the remainder incompound in the flask (table 4).

TABLE 4 Purity after freeze drying. Exp no J1:Rt (%) Rt (%) Rt (%) Run 1J1C1 2.25 (97%) 2.32 (3%) J1C2 2.24 (97%) 1.87 (1%) 1.98 (1%) J1C3 2.22(99%) 1.87 (1%) Run 2 J1C1  2.25 (100%) J1C2 2.25 (95%) 1.88 (3%) 1.98(2%) J1C3 2.25 (97%) 1.87 (3%)

In conclusion, by dissolving J1 in 70% ethanol, diluting with 5 mM HCland freeze-drying three samples were obtained with purity >95%.

Example 1E

It was then tested to omit the acid and instead dilute the ethanol withdeionized water. Three samples of J1 were prepared by dissolving J1 (ca3 mg) in 70% aq. ethanol (0.5 mL) at room temp. The solutions werediluted with deionized water to give a concentration of 0.4 mg/mL. Thesolutions were then frozen in a bath of dry ice-acetone (−78° C.).Freeze-drying was then conducted over 16 h at a pressure of 1 mbar atroom temperature with a dry ice-acetone (−78° C.) trap between thedrying flask and the pump. The visual appearance after drying was assummarized in Table 5 and the purities in Table 6.

TABLE 5 Three replicates of J1 dissolved in ethanol and water. Exp no mgJ1 mL water conc (mg/mL) appearance after drying J1D1 3.15 7.37 0.4white fluffy solid J1D2 3.11 7.27 0.4 white fluffy solid J1D3 3.17 7.420.4 white fluffy solid

TABLE 6 Purity after freeze drying. Exp no J1:Rt (%) J1D1 2.26 (ca 100%)J1D2 2.26 (ca 100%) J1D3 2.25 (ca 100%)

By dissolving J1 in 70% ethanol, diluting with water and freeze-drying;three replicate samples were obtained with the same purity as thestarting material.

Example 2 Effect of Excipients on the Dissolution Rate of LyophilizedMelphalan Flufenamide

In this experiment the effect on speed of dissolution by addingexcipients to the freeze-drying process of melphalan flufenamidehydrochloride (J1) was tested. The following excipients were used, allof which are common formulation agents Generally Considered As Safe(GRAS) according to the FDA (US Food and Drug Administration):

-   -   D-mannitol, trehalose and sucrose;    -   Trizma hydrochloride and L-histidine;    -   Polysorbate 80, β-cyclodextrin;

J1 was used in all experiments.

D-Mannitol, was bought from Sigma no. 33440;

D-(+)-Trehalose dihydrate, was bought from Sigma no. T9449-25 g;

Trizma hydrochloride, was bought from Sigma no. T3253-100 g;

β-Cyclodextrin hydrate, was bought from Sigma no. 856088-5 g;

Polysorbate 80, was bought from Fluka 59924-100 g.

Freeze-drying was performed on a Leybold Lyovac GT2 equipment. LCMS(Liquid chromatography-mass spectrometry) was run on a HP1100-systemusing acetonitrile-0.1% trifluoroacetic acid in water as eluent. AnACE-column C8, 50×3 mm and a gradient 10-97% acetonitrile in 3 min wasused. The filter vials were from Whatman, Mini-UniPrep, 0.45 μm.

(i) Method A, Freeze-Drying

Melphalan flufenamide (30.1 mg) was dissolved in 5 mL of 70% ethanolwith 1 mM HCl, total dissolution within 12 min at 18-19° C. The solutionwas diluted with water (70 mL) and distributed (10 mL) into 250 mL roundbottomed flasks with and without excipient (eg β-cyclodextrin, 9 mg).When all material had dissolved, the solutions were frozen by immersionin a dry-ice/acetone bath at −78° C. The frozen solutions were thenfreeze-dried at <0.1 mbar overnight and room temperature, evaporationkeeping the samples frozen until dryness.

(ii) Method A, Speed of Dissolution Measurement

A 5% glucose solution (10 mL) was added in one portion at 18.5-19° C. tothe freeze-dried material and stirred with a magnet. Aliquots (ca 0.3mL) was taken with 1-mL syringe at various times and filtered through afilter vial (0.45 □m). The filtrate (8 □L) was analyzed by HPLC.

(iii) Method B, Freeze-Drying

Melphalan flufenamide (10.2 mg) was dissolved in 1.67 mL of 70% ethanolwith 5 mM HCl, total dissolution within 5 min at 25° C. The solution wasdiluted with water (23.3 mL) and distributed (10 mL) into flasks withand without excipients (e.g. β-cyclodextrin, 9 mg). The solution of J1and excipient was dispensed into plastic vials with a fitting insert0.45 μm filter (0.25 mL to each vial). The vials were frozen byimmersion in a dry-ice/acetone bath at −78° C. and then kept at −20° C.overnight in a rack fitting the vials. The frozen vials were covered byalumina foil to prevent cross-contamination and kept in the rackprecooled to −20° C., while exposing the rack in a desiccator to <0.1mbar overnight, evaporation keeping the samples frozen until dryness.

(iv) Method B, Speed of Dissolution Measurement

A 5% glucose solution (0.5 mL) was added, which contained an internalstandard (3-methoxybenzoic acid, 0.08 mg/mL). After various times (15s-12 min) the contents of the vials were filtered, the filtrate directlytransferred to glass vials to prevent leaking of undissolved materialinto the filtrate and 8 μL of the filtrate injected into the LCMS.

Determination of Dissolution Speed

In a first approach, Method A, aqueous solutions of J1 with differentadditives were freeze-dried in round bottomed flasks. To each freezedried compound, a glucose solution was added with controlled stirring.Small aliquots were withdrawn with a syringe at specific times andfiltered through a 0.45 μm GHP syringe filter. The degree of dissolutionof J1 in the filtrate was then determined by HPLC. This method was usedwith freeze-dried melphalan flufenamide alone and together withD-mannitol, trehalose, sucrose, Polysorbate 80 and β-cyclodextrin. Theresult of these tests showed that J1 was completely dissolved within 2-4min regardless of excipient (see FIG. 1, no excipients, and FIG. 2, withexcipients. See also Table 7). In fact, the dissolution rate for J1lyophilized with excipients was actually faster than could be measuredusing this method.

TABLE 7 Excipient additions to J1 (4 mg) on freeze-drying, Method A.Ratio J1:additive (mg Number of Freeze dried material J1:mg additive)experiments D-Mannitol 4:2 1 D-Mannitol 4:10 2 Trehalose 4:2 1 Trehalose4:10 2 Sucrose 4:10 1 β-Cyclodextrin 4:9 1 β-Cyclodextrin 4:18 2Polysorbate 80 4:0.05 1 Polysorbate 80 4:0.265 2

To improve the precision and enable measurement of dissolution atshorter intervals, Method B was developed. In this method, aqueoussolutions of melphalan flufenamide and excipients (see Table 2) wereadded to 2 mL plastic vials and freeze-dried. Then a glucose solutionwith internal standard 3-methoxybenzoic acid was added without stirring.After varying times (15 s-6 min) the contents of the vial was filteredwith a 0.45 μm GHP vial insert, the filtrate transferred to a glass vialand the degree of dissolution of melphalan flufenamide hydrochloride(J1) determined by HPLC with internal standard. The lack of stirringmade possible a slower dissolution process, both more clinicallyrelevant and easier to measure the kinetics of.

With this method the dissolution kinetics of freeze-dried J1 could befollowed to complete dissolution after 3-4 min (see FIG. 3, noexcipients and FIG. 4, with excipients, see also Table 8).

TABLE 8 Excipients additions to J1 (4 mg) on freeze-drying, Method B.Ratio J1:additive Freeze dried (mg J1:mg Number of material additive)experiments J1:Mannitol 4:20 1 J1:Trehalose 4:20 1 J1:β-Cyclodextrin 4:91 J1-Polysorbate 4:5 1 J1:Trizma HCL 4:8 1

The speed of dissolution of J1, with and without additives, determinedwith Method A and Method B, are summarized in Table 9.

TABLE 9 Summary of dissolution times of J1 with and without additives,Methods A and B. Ratio J1:addative Freeze dried (mg J1:mg Time (min)Time (min) material additive Method A Method B J1 without <2 3-4additives J1:Trehalose 4:2 <2 J1:Trehalose 4:10 <2 J1:Trehalose 4:200.5-1   J1:Sucrose 4:10 <2 J1:Mannitol 4:2 <2 J1:Mannitol 4:10 <2J1:Mannitol 4:20  0.5-0.75 J1:□ β- 4:9 <2 0.75-1   CyclodextrinJ1:β-Cyclodextrin 4:18 <2 J1:Polysorbate 4:0.05 <2 J1:Polysorbate4:0.265 <2 J1:Polysorbate 4:5 0.25-0.5  J1:Trizma HCL 4:8 >12

Purity and Recovery of J1

A sample of melphalan flufenamide hydrochloride (J1) was dissolved in50% aq. acetonitrile and analyzed immediately with LCMS (Liquidchromatography-mass spectrometry), showing only one peak (>99%). Thepurity of J1 directly after dissolution in 70% ethanol containing 1 mMHCl or 5 mM HCl was found to be ca 97%, with a minor byproduct of ca 3%.The amount of this byproduct increased if the solution was left at roomtemperature.

The results demonstrate that the speed of dissolution of freeze-dried J1in glucose solution with stirring was faster than could be measured(Method A), not enabling the effect of excipient additions to be seen.Using a more clinically relevant Method B without stirring, thedissolution of freeze-dried melphalan flufenamide in glucose solutioncould be followed to completeness after 3-4 minutes. Addition ofexcipients β-cyclodextrin, Polysorbate 80, Mannitol and Trehalose to themelphalan flufenamide solution before freeze-drying all gave completedissolution below 1 minute. The fastest dissolution was given byPolysorbate 80 addition, giving complete dissolution at the firsttime-point 15 seconds.

Example 3 Test of Effect of Concentration of the Excipient Polysorbate80 on the Dissolution Rate of Melphalan Flufenamide

The following was performed to test the amount of the excipientPolysorbate 80 to be added in the freeze-drying process of melphalanflufenamide and to maximize the dissolution rate in a 5% glucosesolution. 0, 10, 50 and 100% weight, in relation to melphalanflufenamide of Polysorbate 80 was used. The experiments were run induplicate.

Melphalan flufenamide hydrochloride (J1) was used in all experiments.The Polysorbate 80 used was bought from Fluka, 59924-100 g.

Freeze-drying was done on a Leybold Lyovac GT2 equipment. LCMS was runon a HP1100-system using acetonitrile-0.1% trifluoroacetic acid in wateras eluent. An ACE-column C8, 50×3 mm and a gradient 10-97% acetonitrilein 3 min was used. The filter vials were from Whatman, Mini-UniPrep,0.45 μm.

General preparation of 2 mg/mL stock solution of melphalan flufenamidebefore freeze-drying was performed as follows:

11.0 mg melphalan flufenamide was suspended in 10 mM solution of HCl inabsolute EtOH (0.5 mL). The mixture was stirred for 30 minutes before0.2 mL water was added. The mixture was stirred for 10 minutes at roomtemperature (clear solution) before it was added to a 0° C. solution ofwater (4.8 mL). 0.25 mL of the solution was transferred to a plasticvial containing 10%, 50% or 100% weight Polysorbate 80. The vial wasshaken, cooled and freeze-dried.

A 5% glucose solution with an internal standard 3-methoxybenzoic acidwas prepared by dissolving 3-methoxybenzoic acid (1.2 mg) in water (15mL). The mixture was stirred for 1 hour before 750 mg of glucose wasadded while stirring. 0.5 mL of the 5% glucose solution was added toeach freeze-dried plastic vial and the mixtures were filtered, atdifferent time-points, transferred to a glass vial and the dissolutionof J1 was determined by HPLC.

Determination of Dissolution Rate

J1 (11 mg) was suspended in EtOH (0.5 mL) and stirred for 30 minutes atroom temperature before water (5 mL) was added. The solution was dividedinto 4 different flasks containing 0%, 10%, 50% or 100% weight (inrelation to J1) of Polysorbate 80. The solutions were transferred to 2mL plastic vials and freeze-dried overnight.

A 5% glucose solution with an internal standard 3-methoxybenzoic acidwas added to each vial without stirring and the mixtures were filteredthrough a 0.45 μm GHP vial insert at different time-points (2-300seconds). The filtrate was immediately transferred to a glass vial toprevent leaking from undissolved material. The amount of dissolved J1relative to the internal standard was determined using HPLC.

Results

The speed of dissolution of J1 (1 mg/mL in 5% glucose solution) with,and without Polysorbate 80, is summarized in Table 10 and depicture inFIG. 5.

TABLE 10 Time to achieve steady state Freeze-dried material dissolution(1 mg/mL) (seconds) J1 without additive 300-600 J1 with 10% Polysorbate80 30-60 J1 with 50% Polysorbate 80 30-60 J1 with 100% Polysorbate 8030-60

Table 10 shows that all samples containing freeze-dried J1 and theexcipient Polysorbate 80 dissolves much faster than J1 freeze-dried inthe absence of excipient. Special attention was devoted to the samplecontaining 10% Polysorbate 80 and the time-points in this experimentwere:

immediate filtration, 2 seconds, 15 seconds, 30 seconds and 5 minutes.In the first time-point where the sample was filtered immediately,approximately 40% was dissolved and after 2 seconds approximately 70%was dissolved. Full dissolution was achieved after 30-60 seconds.

The dissolution rate of freeze-dried J1 at 1 mg/mL containing varyingamounts of Polysorbate 80 in a 5% glucose solution was under 1 minutefor all samples. The lowest amount of Polysorbate 80 for rapiddissolution was between 10 and 50% by weight.

Example 4 Test of Effect of Concentrations of the Excipients Polysorbate80, PEG 400 and β-Cyclodextrin on the Dissolution Rate of MelphalanFlufenamide

This example was performed to study the effect of differentconcentrations of the excipients Polysorbate 80, PEG 400 andβ-cyclodextrin added in the freeze-drying process of melphalanflufenamide to maximize the solubility and speed of dissolution in a 5%glucose solution towards the long-term goal of developing a lyophilizedmaterial, stable to storage and with facile preparation for dosing.

Melphalan flufenamide hydrochloride (J1) was used in all experiments.

Polysorbate 80 used was bought from Fluka (59924-100 g), β-cyclodextrinfrom Aldrich (856088) and PEG 400 from Clariant (100316).

Freeze-drying was done on a Leybold Lyovac GT2 equipment. LCMS was runon a HP1100-system using acetonitrile-0.1% trifluoroacetic acid in wateras eluent. An ACE-column C8, 50×3 mm and a gradient 10-97% acetonitrilein 3 min was used. The filter vials were from Whatman, Mini-UniPrep,0.45 μm.

General Preparation of 2 mg/mL Stock Solution of Melphalan Flufenamidefor Freeze Drying

11.1 mg melphalan flufenamide was suspended in 10 mM solution of HCl inabsolute EtOH (0.5 mL). The mixture was stirred for 30 minutes before0.2 mL water was added. The mixture was stirred for 10 minutes at roomtemperature (clear solution) before it was added dropwise to a 0° C.solution of water (4.8 mL). 0.25 mL or 0.5 mL of the solution wastransferred to a plastic vial containing the excipients. The vial wasshaken, cooled and freeze-dried.

Solubility Experiment

A 5% glucose solution with an internal standard was prepared bydissolving 3-methoxybenzoic acid (1.2 mg) in water (15 mL). The mixturewas stirred for 1 hour before 750 mg of glucose was added whilestirring. 0.2 mL of the 5% glucose solution was added to eachfreeze-dried plastic vial and the mixtures were shaken for 10-15 secondsand filtered after 5 minutes. The filtrate was transferred to a glassvial and the solubility of melphalan flufenamide was determined by HPLCand a calibration curve.

Determination of Solubility

A 2 mg/mL stock solution of melphalan flufenamide hydrochloride (J1) wasused as in previous experiments.

For a solubility of 2.5 mg/mL of J1 in 5% glucose solution, 0.25 mL ofthe stock solution was dispensed into 2 mL plastic vials containing amixture of the excipients determined by experimental design and themixtures were immediately cooled and freeze-dried.

The high/low levels of each excipient (in weight-% relative to melphalanflufenamide) were as follows: Polysorbate 80 (8%-80%), PEG 400(80%-400%) and β-cyclodextrin (10%-50%). The highest amount of eachexcipient was determined from FDA Inactive Ingredient database ofregistered IV-administered drugs. β-cyclodextrin is on FDA's GRAS(Generally Recognized As Safe) list but no recommendations are given forintravenous injections to our knowledge, which caused a fairlyconservative high level to be set. The weight percent of each excipientin relation to melphalan flufenamide hydrochloride (J1) (weight) isshown in Table 11.

TABLE 11 Weight percentage of each excipient in relation to J1.Experiment Polysorbate 80 PEG 400 β-cyclodextrin No [%] [%] [%] 1 8 40010 2 8 80 10 3 8 400 50 4 8 80 50 5 80 400 10 6 80 80 10 7 80 400 50 880 80 50 9 44 240 30 10 44 240 30 11 44 240 30

As is demonstrated in other experiments herein, the dissolution rate ofJ1 increased markedly with the addition of Polysorbate 80 in the freezedrying process. Three experiments were performed to attempting to reacha solubility of 5 mg/mL. A stock solution of J1 was added to 3 differentplastic vials (exp 12, 13 and 14) containing Polysorbate 80 (10%, 50%and 100% weight in relation to melphalan flufenamide). The mixtures wereimmediately cooled and freeze-dried.

A 5% glucose solution with an internal standard (3-methoxybenzoic acid)was added to each vial and the vials were shaken and allowed to standfor 5 minutes. The mixtures were filtered through a 0.45 μm GHP filtervial and the filtrate was immediately transferred to a glass vial toprevent leaking from undissolved material. The amount of dissolved J1was determined using HPLC and a calibration curve.

Results

The solubilities of J1 in mg/mL with high/low levels of the excipientsPolysorbate 80, PEG 400 and β-cyclodextrin are summarized in Table 12.

TABLE 12 Solubility of J1 in mg/mL. Polysorbate 80 PEG 400β-cyclodextrin Solubility of Experiment [%] [%] [%] J1 [mg/ml] 1 8 40010 1.9 2 8 80 10 0.9 3 8 400 50 2 4 8 80 50 1.4 5 80 400 10 2 6 80 80 101.5 7 80 400 50 2 8 80 80 50 1.9 9 44 240 30 1.9 10  44 240 30 1.8 11 44 240 30 1.7 12  10 x x 1 13  50 x x 1.2 14  100 x x 1.4 15* x x x 0.6716* x x x 0.25 17  50 80 100 1.2 *Experiments 15-16 did not usefreeze-dried J1, fine powder was used in Experiment 15 and larger lumpswere used in Experiment 16.

The results provided in Table 12 demonstrates that the solubility of J1increased in all experiments containing excipients compared to notfreeze-dried J1 (entry 15 and 16).

The large discrepancy in the solubility of not freeze-dried J1 isprobably due to different particle size in the batch, since entry 15 wasa suspension of fine white powder, while entry 16 was larger lumpsgiving lower dissolution rate and hence lower solubility of J1 in 5minutes. The precision of the analysis is shown in center experiments9-11 (1.9, 1.8 and 1.7) with identical excipient concentrations. The 3samples with Polysorbate 80 as the excipient (10, 50 and 100%) exhibiteda solubility of 1.0, 1.2 and 1.4 mg/mL, respectively.

The entries with a mixture of the excipients Polysorbate 80, Peg 400 andβ-cyclodextrin exhibited several combinations with solubilities at orclose to 2.0 mg/mL. The highest determined solubilities 2.0 (entries 3,5 and 7) were only attainable with high levels of PEG 400, givingliquids or semisolids after freeze drying.

The samples with lower amount of PEG 400 (entries 2, 4, 6 and 8) formeda white fluffy powder after freeze drying, with the highest determinedsolubility of 1.9 mg/mL in entry 8. This prompted testing if a highersolubility could be obtained by lowering the amount of PEG 400 andincreasing the amount of β-cyclodextrin. An additional sample (row 17 inTable 12) was freeze-dried containing 50% Polysorbate 80, 80% of PEG 400and 100% of β-cyclodextrin. The solubility of J1 with this mixture ofexcipients was 1.2 mg/mL.

The results demonstrate that the maximum solubility of J1 withcombinations of excipients is close to 2 mg/mL.

With experiment 13 it was shown that a solution of J1 with 50%Polysorbate gave a solubility of approximately 1.2 mg/mL alone,sufficient for a 1.0 mg/mL formulation and allowing the exclusion of PEG400 and β-cyclodextrin.

Visual Confirmation Experiments

To confirm the dissolution in a more clinically relevant setting, alarger scale experiment in transparent glass vials instead of plasticvials was performed. Vial 1 contained a solution 4.8 mg melphalanflufenamide hydrochloride (J1) and 2.4 mg Polysorbate 80. As a controlvial 2 contained 4.8 mg melphalan flufenamide hydrochloride (J1) and noPolysorbate 80. The vials were freeze-dried overnight.

To each vial containing the freeze-dried melphalan J1 as white fluffymaterial, 4.70 mL of a 5% glucose solution was added to give aconcentration of J1 of 1.02 mg/mL. The mixtures were shaken for 10-15seconds and the test tube containing J1 and 50% Polysorbate 80 showed aclear solution after 15 seconds, see FIG. 6, left vial. The referencetube with freeze-dried J1 without the Polysorbate showed small particlesand was not totally dissolved after 30 minutes, see FIG. 6, right vial.LC-MS analysis revealed that the purity of melphalan flufenamide after30 minutes was >95%, in both vials.

The results provided herein demonstrate that the solubility of J1 in 5%glucose solution could be enhanced using a mixture of the excipientsPolysorbate 80, Peg 400 and β-cyclodextrin to 1.9 mg/mL. Such a mixtureof excipients with J1 resulted in a fluffy white solid onlyophilization.

Lyophilization of J1 with 50%-weight Polysorbate 80, resulted in a whitefluffy solid that is rapidly dissolved in 5% glucose solution. Thesaturation concentration 1.2 mg/mL is sufficient to use in a clinicalsetting for dosing preparation at 1.0 mg/mL.

Example 5 Stability Test

The purpose of the first part of this study was to investigate thedissolution rate of melphalan flufenamide hydrochloride (J1)(freeze-dried together with Polysorbate 80) in 5% glucose solution.

The dissolution speed of J1 (freeze-dried) in 5% glucose solutioncontaining Polysorbate 80 will be measured in another experiment.

Finally the dissolution speed of non-freeze dried J1 in 5% glucosesolution containing Polysorbate 80 will be measured.

The second part is an investigation of the degradation of J1 in twodifferent preparations at elevated temperature. The first preparationwas a freeze dried solid containing polysorbate 80 and the second was a25 mg/ml solution of J1 in N,N-dimethylacetamide (DMA). The degradationwas followed for 1 month at +40° C., using two preparations

(i) Determination of Dissolution Rate.

A 5% glucose solution was added to each plastic vial containing J1. Thevials were were shaken and filtered at different time-points. Thefiltrate was transferred to glass vials and the amount of dissolved J1was determined by HPLC.

(ii) Accelerated Stability Study Design.

Vials with freeze-dried J1 and Polysorbate 80, and 10 vials of J1solution in DMA, were stored at 40° C. for 1 month. Two vials of thefreeze-dried material (named freeze dried 1 and 2 in table below) andone vial of the DMA solution (named DMA in table 1 below) was taken outfrom the 40° C. chamber and stored at −20° C. and analysed at the sametime for assay and purity of J1. Sample times were 0, 1, 3, 10 and 30days. Each freeze dried vial contained 0.25 mg of J1. The 25 mg/mlsolution in DMA was from Oncopeptides.

(iii) Analysis and Results

The freeze dried samples were dissolved in 500 μl DMA in Whatman 0.45 μmfilter vials. The samples were vortexed briefly before pressing the twoparts of the vial together and thus filtrating the sample. The 25 mg/mlsolution samples were diluted with DMA by aliquoting 20 μl solution toHPLC vials and diluting with 980 μl DMA. 4 μl were injected in thechromatographic system.

The stability was evaluated as the relative purity, since there was aslight variation in the amount of J1 in the freeze dried vials. By usingrelative purity, each sample is standardised against itself and theeffect of varying J1 amount is minimised on the stability result.

The speed of dissolution of J1 in 5% glucose in the presence of PS issummarized in table 13:

TABLE 13 Summary of dissolution experiments. Time (min) to reach steadystate Content in plastic dissolution vial Solution Dissolution 1freeze-dried 5% glucose exp. 1 J1 + Polysorbate 80 Dissolution 1freeze-dried J1 5% glucose + exp. 2 Polysorbate 80 Dissolution 1-2non-freeze-dried J1 5% glucose + exp. 3 Polysorbate 80

Stability Test Results

TABLE 14 Results of stability test at 40° C., comparison between DMAsolution and freeze-dried J1 at +40° C./Ambient Relative Humidity Freezedried 2 Freeze dried 2 Freeze dried Avg DMA Day Relative [%] Relative[%] Relative [%] Relative [%] 0 98.80 98.74 98.77 96.81 1 98.77 98.6998.73 95.76 3 98.71 98.77 98.74 95.43 10 98.55 98.66 98.61 92.22 3098.32 98.42 98.37 86.90

The results in table 14 show that the freeze dried material isessentially unchanged during the test period. Only a small change inpurity can be observed. Also the dissolution rate of freeze-dried J1 at1 mg/mL in a 5% glucose solution was under 1 min in the presence ofPolysorbate 80. The dissolution rate of non-freeze-dried J1 at 1 mg/mLin a 5% glucose solution containing Polysorbate 80 was estimated to 1-2min.

J1 in DMA solution degraded significantly during storage at +40° C. forone month. The relative amount decreased from about 96.8% to 86.9%. J1stored as a freeze dried solid only showed a small degradation from98.7% to 98.3% during the same period of time.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-40. (canceled)
 41. A lyophilized pharmaceutical preparation comprisingmelphalan flufenamide hydrochloride (J1) and sucrose.
 42. Thelyophilized pharmaceutical preparation according to claim 41, whereinthe amount of sucrose is 10-100% by weight of said melphalan flufenamidehydrochloride (J1).
 43. The lyophilized pharmaceutical preparationaccording to claim 41, which is free, or substantially free from organicsolvents.
 44. The lyophilized pharmaceutical preparation according toclaim 42, which is free, or substantially free from organic solvents.45. A pharmaceutical composition consisting of the lyophilizedpharmaceutical preparation of claim 41 and a physiologically acceptablesolution, wherein said physiologically acceptable solution is a glucosesolution.
 46. A pharmaceutical composition consisting of the lyophilizedpharmaceutical preparation of claim 42 and a physiologically acceptablesolution, wherein said physiologically acceptable solution is a glucosesolution.
 47. A pharmaceutical composition consisting of the lyophilizedpharmaceutical preparation of claim 43 and a physiologically acceptablesolution, wherein said physiologically acceptable solution is a glucosesolution.
 48. A pharmaceutical composition consisting of the lyophilizedpharmaceutical preparation of claim 44 and a physiologically acceptablesolution, wherein said physiologically acceptable solution is a glucosesolution.
 49. A method for treating and/or preventing cancer, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the lyophilized pharmaceutical preparation of claim
 41. 50. Amethod for treating and/or preventing cancer, comprising administeringto a subject in need thereof a therapeutically effective amount of thelyophilized pharmaceutical preparation of claim
 42. 51. A method fortreating and/or preventing cancer, comprising administering to a subjectin need thereof a therapeutically effective amount of the lyophilizedpharmaceutical preparation of claim
 43. 52. A method for treating and/orpreventing cancer, comprising administering to a subject in need thereofa therapeutically effective amount of the lyophilized pharmaceuticalpreparation of claim
 44. 53. The method of claim 49, wherein said canceris ovarian cancer, lung cancer, bladder cancer, mesothelioma, multiplemyeloma, breast cancer or hematological cancer.
 54. The method of claim50, wherein said cancer is ovarian cancer, lung cancer, bladder cancer,mesothelioma, multiple myeloma, breast cancer or hematological cancer.55. The method of claim 51, wherein said cancer is ovarian cancer, lungcancer, bladder cancer, mesothelioma, multiple myeloma, breast cancer orhematological cancer.
 56. The method of claim 52, wherein said cancer isovarian cancer, lung cancer, bladder cancer, mesothelioma, multiplemyeloma, breast cancer or hematological cancer.
 57. A method forpreparing the lyophilized pharmaceutical preparation of claim 41,comprising: (a) dissolving melphalan flufenamide hydrochloride (J1) inan organic solvent to obtain a melphalan flufenamide hydrochloride (J1)solution; (b) combining the melphalan flufenamide hydrochloride (J1)solution with water to obtain an aqueous melphalan flufenamidehydrochloride (J1) solution, wherein the concentration of the melphalanflufenamide hydrochloride in the solution ranges from 0.2-3.0 mg/ml; (c)combining the melphalan flufenamide hydrochloride (J1) solution withsucrose to obtain a melphalan flufenamide hydrochloride (J1) solutioncontaining sucrose; and (d) lyophilizing the aqueous melphalanflufenamide hydrochloride (J1) solution containing sucrose.
 58. Themethod of claim 57, wherein the organic solvent is selected from thegroup consisting of ethanol, ethanol containing acid, glycerin,propylene glycol, benzyl alcohol, dimethylacetamide (DMA),N-methyl-2-pyrrolidone, isopropanol, n-butanol, tert-butanol, methyltert-butyl ether, propylene glycol, dimethylsulfoxide, tetrahydrofuran,2-methyl tetrahydrofuran, acetone, dimethylformamide, acetonitrile,dioxane, acetic acid, lactic acid, propionic acid, n-butanol,isopropanol, n-propanol, tert-butanol, sec-butanol, methanol, and amixture of ethanol and water.
 59. A method for decreasing thereconstitution time of a lyophilized preparation of melphalanflufenamide hydrochloride (J1) when reconstituted in an aqueous solvent,comprising lyophilizing melphalan flufenamide hydrochloride (J1) in thepresence of sucrose.
 60. The method of claim 59, which comprisesdissolving the melphalan flufenamide hydrochloride (J1) in ethanol toobtain a melphalan flufenamide hydrochloride (J1) solution and combiningthe solution with sucrose prior to lyophilization.